The Pancreas Duodenum Homeobox-1 protein (Pdx1) is one of the most important transcriptional regulators in the pancreas, since it plays a fundamental role in the early formation of all pancreatic cell types and in the production and function of adult islet ? cells. Moreover, inactivation of human Pdx1 contributes to diabetic islet ? cell dysfunction. Gene regulation by transcription factors (TFs) like Pdx1 necessitates the recruitment through protein-protein interactions of coregulators, which confer a second level of specificity to the transcriptional response due to their positive and/or negative actions. Because little was known about the coregulators recruited by islet-enriched TFs, a principal objective during the prior funding cycle was to isolate coregulators of Pdx1 from mouse ? cells. While many were identified, we focused on determining the significance to Pdx1 of the Swi/Snf chromatin remodeling complex. Our studies not only strongly suggest that Swi/Snf is a critical regulator in ? cell lines, but that recruitment to Pdx1 is compromised under the dysfunctional conditions associated with human Type 2 diabetes. A major objective in this proposal will be to determine the in vivo significance of Pdx1:Swi/Snf control in the developing pancreas and islet ? cells postnatally, with preliminary results supporting a prominent role. Because Swi/Snf does not have as powerful a regulatory impact as Pdx1, we will also work towards defining the underlying transcriptional mechanisms of other Pdx1 recruited coregulators on islet ? cell activity. Our earlier transgenic- and cell line-based experiments localized pancreatic cell-type-specific transcriptional regulatory areas of the Pdx1 gene to conserved 5'-flanking region sequences. We have constructed deletion mutants in the Area IV (base pair (bp) -6200/-5670) and Area II (bp -2153/-1923) control regions of the endogenous mouse Pdx1 gene to analyze the individual contributions of these enhancers to pancreatic cell type specification, differentiation, and function in vivo. Strikingly, the molecular and physiological impact of the Pdx1?Area IV mutant was almost exclusively during the postnatal weaning period. Another principal focus will be on determining if islet ? cell dysfunction in this mutant disrupts the activity of the circadian clock, as the mediating CLOCK and BMAL1 TFs reside within Pdx1 bound transcriptionally active enhancers and yield a similar in vivo TF mutant phenotype to Pdx1?Area IV. Collectively, our studies will focus on defining mechanisms used by Pdx1 in controlling the normal formation and activity of islet ? cells, and determining their possible contribution in the pathogenesis of diabetes. These findings will have a significant positive impact by providing knowledge relevant to those developing diagnostic and therapeutic approaches for disease treatment.
Pdx1 is viewed as a master regulator in the pancreas because of its essential role in early pancreas formation and subsequently in islet ? cells. Here we define mechanistically how transcriptional coregulators recruited by Pdx1 influences these processes. In addition, we will determine how Pdx1 affects the circadian clock within mouse islet ? cells at weaning, specifically by addressing how this transcription factor affects enhancer binding of key regulators of the circadian response, BMAL1 and CLOCK.
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